Graft versus host disease (GVHD), which is a disease that is caused after bone marrow transplantation and damages many organs; hemophagocytic syndrome (HPS), which is a disease where blood cells are phagocytized by phagocytes, especially virus associated hemophagocytic syndrome (VAHS) occurring after viral infection; and so forth are severe diseases that cause organ failures and eventually lead to death. Currently, cytologic diagnosis such as biopsy is generally used for diagnosing these diseases. However, such diagnosis suffers from drawbacks in view of patient's pain during the test, time required for diagnosis and operation procedures.
Since it has been found in recent years that a major cause of GVHD and HPS is cell apoptosis in living bodies, it is considered that GVHD and HPS may be diagnosed by detecting apoptosis.
As methods for detecting apoptosis, there have conventionally been used 1) morphologic methods, 2) histochemical methods, 3) biochemical methods and 4) immunochemical methods.
1) Morphologic Methods
There are used the chromatin codensation method, in which DNA fragmentation specifically occurring during apoptosis is detected by staining, and a method of detecting morphologic change specific to apoptosis by observation under an electron microscope or measurement of a cell size.
2) Histochemical Methods
There is used a method of ligating a labeled nucleotide to a terminus of a DNA fragment and detecting it by a fluorescence microscope. As another method, a flow cytometry is also used, in which amounts of intracellular substances are measured for individual cells.
3) Biochemical Methods
There is used a method of detecting a DNA ladder by agarose gel electrophoresis. This is currently considered to be the most reliable method for identifying apoptosis. However, since DNA is extracted from a tissue or a cell population as a whole, it is said that this method has a problem in sensitivity and quantitation performance when the proportion of apoptotic cells is not high.
4) Immunochemical Methods
A solid phase enzyme immunoassay (enzyme-linked immunosorbent assay, ELISA) for detecting histone-binding DNA fragments (mono- or oligo-nucleosome) has been developed (Cell Detection ELISA: Boehringer Mannheim, Kokusan Chemical).
However, these methods have problems such as complicated procedures and poor sensitivity and quantification performance, and have not been currently used in practice as methods for diagnosing GVHD and HPS.